1. Field of the Invention
The present invention relates to a viral vector, especially relates to a viral vector whose transcripts induce gene inactivation in plants.
2. The Prior Arts
Orchidaceous is one of the important plants with economic values. As for drugs derived from some orchids, more of them become important ornamental plants because of their enchanting flowers or showy leaves. Extensive breeding have also been conducted in Phalaenopsis spp. and more than 18,000 hybrids have been bred. Diversified flowers appearance may provide resources for elucidating evolution of genes involved in floral morphogenesis and their related function.
Methods to understand and analyze plant gene function are employed by using loss-of-function or gain-of-function techniques at present. Gene function analyzed by gain-of-function is generally accomplished through gene transformation, while loss-of-function is conducted through mutagen, transposon tagging, T-DNA insertion or homologous recombination. However, the abovementioned approaches are complicated, time-consuming and difficult to scale up the gene analysis. And methods for gene transformation are not established in many orchid species, such as Phalaenopsis spp., because of their lengthy life cycle. Two years are needed for flowering either with seed propagation or tissue culture after sowing. Therefore it is very time-consuming and unrealistic to analyze genes involved in floral development. In addition, genome sizes among various Phalaenopsis spp. are quite large, ranging from 1×109 to 6×109 bp/1C, and the multiploidy of commercial cultivars makes the genetic analysis more complicated.
Alternatively, gene silencing is used to analyze gene function recently. Virus induced gene silencing (VIGS) is an efficient and reliable method though there are many techniques being conducted to induce gene silencing. Gene transformation is not required in the VIGS process, which provides a quick and feasible way for plants with lengthy life cycle and transformation difficulties. The exact mechanism of VIGS remains unknown, so far it's been postulated that VIGS is related to host defense mechanisms to defend against foreign viral RNAs or transposon(s). Small interfering RNA (siRNA) has been observed in plants when foreign viruses invade plants. These siRNAs become, hybridize foreign viral RNAs and trigger nuclease to degrade viral RNA. Virus-induced gene silencing (VIGS) is a type of RNA silencing that is initiated by virus vectors carrying host genes being analyzed. The plants are infected with the transcripts to activate the RNA silencing of endogenous gene of host plants.
VIGS does not require the whole gene. It was reported that 23 nucleotides was enough to induce gene silencing (Plant J. 25, 417-25, 2001). This will alleviate problems of acquiring the whole cDNA. Compared with transformation of plants with sense and/or antisense gene approaches, the advantage of virus induced gene silencing (VIGS) is the relative speed. Moreover it suppresses the target gene RNA level after the seedling established, which will prevent suppression of some essential genes needed in development stages.
Kumagai et al. inserted partial phytoene desaturase (PDS) gene fragment into tobacco mosaic virus (TMV) cDNA clone (Proc. Natl. Acad. Sci. U.S.A. 92, 1679-83, 1995). In vitro transcripts derived from the infectious clone were used to inoculate plants. Initially infected plants show typical symptoms of TMV infection. One or two weeks later endogenous PDS mRNA could not be detected, and plants produced white leaves caused by decreasing of PDS gene expression. Ruiz et al. reported similar results using a potato virus X (PVX) based vector (Plant Cell, 10, 937-46, 1998). Burton et al. utilized a PVX vector to silence plant cellulose gene (Plant Cell, 12, 697-705, 2000). Most of the VIGS vectors constructed are applied in dicots. Among monocots, only Barley strip mosaic virus derived vectors have been developed by Holzberg et al. for VIGS (Plant J., 30, 315-27, 2002).
Orchid plants are monocots of economically importance, which own a large market. So far there is no report regarding the application of VIGS vectors in orchids. The gene function of orchid plants can be studied if a VIGS vector is developed to be applied in orchid plants. VIGS vectors help to screen genes of commercial values rapidly, especially towards flower morphology genes, as well as genes involved in related traits, which will have high commercial values.